International Journal of Molecular Sciences Article Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro Egor A. Turovsky 1,2,* , Maria V. Turovskaya 1, Evgeniya I. Fedotova 1, Alexey A. Babaev 2, Victor S. Tarabykin 2,3 and Elena G. Varlamova 1,* 1 Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, Institute of Cell Biophysics of the Russian Academy of Sciences, 142290 Pushchino, Russia; [email protected] (M.V.T.); [email protected] (E.I.F.) 2 Institute of Neuroscience, Lobachevsky State University of Nizhniy Novgorod, 23 Prospekt Gagarina, 603950 Nizhny Novgorod, Russia; [email protected] (A.A.B.); [email protected] (V.S.T.) 3 Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin, Berlin, Charitéplatz 1, 10117 Berlin, Germany * Correspondence: [email protected] or [email protected] (E.A.T.); [email protected] or [email protected] (E.G.V.) Abstract: Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurode- generative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription Citation: Turovsky, E.A.; Turovskaya, factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and M.V.; Fedotova, E.I.; Babaev, A.A.; KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Tarabykin, V.S.; Varlamova, E.G. Role Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, of Satb1 and Satb2 Transcription thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient Factors in the Glutamate Receptors (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist Expression and Ca2+ Signaling in the reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of Cortical Neurons In Vitro. Int. J. Mol. 2+ Sci. 2021, 22, 5968. https://doi.org/ the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca -signals of neurons 10.3390/ijms22115968 in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of Academic Editors: Elek Molnár and the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion Julie Perroy decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory Received: 12 May 2021 system of neurotransmission resulting in the suppression of the neuron maturation process and Accepted: 28 May 2021 switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a Published: 31 May 2021 similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal Publisher’s Note: MDPI stays neutral network in the cortical cell culture. with regard to jurisdictional claims in published maps and institutional affil- Keywords: Satb1; Satb2; calcium; glutamate receptors; GABA-receptors; gene deletion; neurons; iations. cortex; signal transduction Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article Neuronal transcription factors regulate the expression of many receptors and intracel- distributed under the terms and lular signaling molecules involved in excitatory neurotransmission [1,2]. Great attention conditions of the Creative Commons has been paid to transcription factors Satb1 and Satb2 in terms of their contribution to 2+ Attribution (CC BY) license (https:// embryonic brain development. However, the effect of these factors on the Ca -signaling creativecommons.org/licenses/by/ of neurons has not been practically investigated. The special AT-rich sequence-binding pro- 4.0/). teins 1 and 2 (Satb1/2) are nuclear matrix-associated proteins that exert multiple functions Int. J. Mol. Sci. 2021, 22, 5968. https://doi.org/10.3390/ijms22115968 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5968 2 of 31 by influencing the structural organization of chromatin and interacting with several co- activators and co-repressors of transcription. Satb1 was first cloned from a cDNA library of the human cell line and found in thymocytes [3]. Further, this factor was discovered in other tissues and cells. Satb1 ((MIM) 602075) gene resides on chromosome 3p24.3. The protein consists of 763 amino acids (~86 kDa molecular weight, migrates as 103 kDa on SDS gels) and includes six domains: nuclear localization signal, PDZ, BUR-binding domain, two Cut repeats (CUT1 and CUT2), and homeodomain (HD). Satb1 functions as a highly pleiotropic regulator of gene expression which dynamically alters the organization and epigenetic status of the chromatin [4]. Changes in Satb1 expression are associated with human tumors, including rectal cancer, cutaneous malignant melanoma, breast and prostate epithelia [5–7]. Satb1 is associated with highly metastatic breast cancer [4]. Most of the Satb1-regulated genes such as errb2, abl1, mmp2, e-cadherin, vegfb, tgfb1, and kiss1 play an important role in the induction of carcinogenesis [8,9]. Satb1 via MAR-mediated interactions with DNA- regulatory elements is a transcriptional repressor [10]. Moreover, Satb1 may function as a “docking site” for several chromatin-modifier proteins that suppress gene expression [11]. Satb1 is more abundant in the brain and expressed exclusively in the neurons. Significant expression is observed in the neocortex, nucleus of the diagonal band, amygdala, and tegmental area. In contrast, in the case of the ventral midbrain, Satb1-positive neurons were observed only in the small part of the substantia nigra, ventral tegmental area, and retrorubral field, but no Satb1-positive neurons were found in the inferior colliculus. Thus, Satb1-positive neurons were predominantly dopaminergic [12]. The transcription factor Satb1 is extensively expressed in SST+-, CR+-, and NPY+-interneurons, while the expression was not observed in VIP+-interneurons [13,14]. Mice with Satb1 mutation are characterized by incomplete eye-opening and the clasping reflex [15]. The Satb2 ((MIM) 608148) gene localizes in chromosome 2q32–q33, spans 191 kb, and contains 11 exons. Its open reading frame begins in exon 2, with the first stop codon in exon 11, predicting a 733 amino acid protein. The protein contains a Pfam-B_10016 domain required for dimerization (residues 57–231), two CUT domains (352–437 and 482–560), and a homeodomain (614–677) [16]. Satb2 is highly conserved (99.6%) among the members of the transcription-factor gene families which members bind to nuclear matrix attachment regions and appear to be involved in the regulation of the tissue-specific organization of chromatin [17,18]. Furthermore, Satb2 is a target for SUMOylation—a reversible modification of the protein that modulates its activity [18]. A single case of a de novo nonsense mutation in Satb2 has been described in individuals with cleft palate, osteoporosis, profound mental retardation, epilepsy, a jovial personality, and craniofacial dysmorphism including gum hyperplasia, mandibular hypoplasia, and anterior pointing incisors [19]. Satb2 is a genetic risk locus for schizophrenia and a regulator of several miRNAs responsible for the translation of proteins that control certain aspects of synaptic plasticity and memory formation: miR-124, miR-125b, miR-132, miR-212, miR-381, miR- 326, and miR-19b [20,21]. Alterations in the expression and activity of Satb2 cause Satb2- associated syndrome in humans. SAS (OMIM 612313) is a clinically recognizable syndrome characterized by neurodevelopmental and behavioral abnormalities, palatal and dental anomalies, dysmorphic features, and frequent skeletal pathology [22–24]. Patients with SAS are characterized by deep mental retardation, and learning difficulties [22,25]. Both of these transcription factors are involved in the regulation of the growth and development of various types of brain neurons, both in the embryonic and postnatal periods [17]. We have previously shown that mutations of the Sip1 or MeCP2 transcription factor cause a disturbance of Ca2+-signaling in neurons and astrocytes [26,27]. Changes in the expression level and activity of glutamate receptors upon deletion of transcription factors Satb1 and Satb2 may be considered one of the key fundamental mechanisms that determine the triggering of the neural network to a hyperexcitation state. Despite a sufficient number of works devoted to the role of Satb1 and Satb2 in neurogenesis and brain development, there are no studies demonstrating the changes in intracellular calcium signaling when these transcription factors are impaired in neurons. In our paper, Int. J. Mol. Sci. 2021, 22, 5968 3 of 31 we found that the expression of Satb1 and Satb2 is necessary for the development of excita- tory and inhibitory neurotransmission, and conditional knockout of these